The long-term objective of this research is to understand the molecular mechanism by which stretching a muscle makes it pull harder. All striated muscles contract via myosin motor proteins that cyclically pull on actin filaments. In a relaxed muscle, myosin is sterically blocked from interacting with actin by the troponin- tropomyosin complex. In a neurogenic contraction, troponin binds released calcium, which relieves the steric blocking by tropomyosin and enables myosin to bind to and pull on actin. By an unknown mechanism, stretching a muscle makes it pull harder, a process known as length dependent activation if pre-stretching a relaxed muscle leads to greater force in subsequent contractions, or as stretch activation if stretching a partially activated muscle leads to a delayed rise in force. Length dependent activation is an essential feature of cardiac muscle underlying the Frank-Starling Law of the Heart, which allows it to match the input and output volumes beat-to-beat by increasing systolic force after increased diastolic filling. Stretch activation is an essential feature of most insect flight muscles, which allows it to mechanically trigger contraction during flight. All striated muscles, vertebrate or insect, show varying degrees of length dependent activation and stretch activation, but it is currently not known whether these two processes reflect the same underlying phenomenon, nor is the molecular mechanism for either known, despite decades of research. Current data from insect flight muscle suggest that stretch activation is controlled by tropomyosin, similar to neurogenic calcium-activation but mediated by myosin-troponin connections that transmit strain when the muscle is stretched. This project is the first ever systematic comparison of length dependent activation and stretch activation in vertebrate cardiac, slow skeletal, fast skeletal, and insect flight muscles, to determine whether the two modes of activation are different manifestations of a single process for all muscle types. Preliminary data from bovine cardiac muscle and insect flight muscle suggest that length dependent activation and stretch activation are a single process. Troponin-exchange among all four muscle types will determine the type-specific requirements for length dependent activation and stretch activation as judged by the physiological responses, and for myosin-troponin connections as judged by electron microscopy. Real-time X-ray diffraction movies of stretch activation in rabbit psoas muscle will elucidate the molecular mechanisms of stretch activation and length dependent activation in vertebrate striated muscle by revealing the sequence of molecular structural changes, to be compared side by side with insect flight muscle. Understanding the molecular basis of length dependent activation, stretch activation, and the action of myosin-troponin-bridges is necessary for a detailed mechanistic understanding of normal muscle function, which in turn is an essential prerequisite for understanding how these mechanisms are deficient in human disease, including heart disease, muscle myopathies, muscle injuries, and sarcopenia. PUBLIC HEALTH RELEVANCE: This project seeks to understand the molecular mechanism by which pulling on a muscle makes it pull back harder. This response is especially well developed in heart muscle where it modulates the force of contraction beat by beat. A deeper understanding of this process may lead to better prevention of and treatment for heart disease.